Known Methods of Treatment
It has been known that malignant tumors result from the uncontrolled growth of cells in an organ. The tumors grow to an extent where normal organ function may be critically impaired by tumor invasion, replacement of functioning tissue, competition for essential resources and, frequently, metastatic spread to secondary sites. Malignant cancer is the second leading cause of mortality in the United States.
Up to the present, the methods for treating malignant tumors include surgical resection, radiation and/or chemotherapy. However, numerous malignancies respond poorly to all traditionally available treatment options and there are serious adverse side effects to the known and practiced methods. There has been much advancement to reduce the severity of the side effects while increasing the efficiency of commonly practiced treatment regimens. However, many problems remain, and there is a need to search for alternative modalities of treatment. The search is particularly urgent for primary malignant tumors of the central nervous system. Brain tumors, especially glioblastomas, remain one of the most difficult therapeutic challenges. Despite the application of surgery, radiotherapy and chemotherapy, alone and in combination, glioblastomas are almost always fatal, with a median survival rate of less than a year and 5-year survival rates of 5.5% or less. None of the available therapeutic modes has substantially changed the relentless progress of glioblastomas.
Systematic studies of patients who were diagnosed with malignant glioma and underwent surgery to wholly or partially remove the tumor with subsequent chemotherapy and/or radiation showed that the survival rate after 1 year remains very low, particularly for patients who are over 60 ears of age. Leibel, S. A., et al., Cancer, 35:1551–1557 (1975); Walker, M. D., et al., J. Neurosurg., 49:333–343 (1978); Chang, C. H., et al., Cancer, 52:997–1007 (1983). Malignant gliomas have proven to be relatively resistant to radiation and chemotherapeutic regimens. Bloom, H. J. G., Int. J. Radiat. Oncol. Biol. Phys., 8:1083–1087 (1982). Adding to the poor prognosis for malignant gliomas is the frequent tendency for local recurrence after surgical ablation and adjunct radiation/chemotherapy. Choucair, A. K., et al., J. Neurosurg., 65:654–658 (1986).
Treatment of Cancer with Viruses
In recent years, there have been proposals to use viruses for the treatment of cancer: (1) as gene delivery vehicles, Miller, A. D., Nature, 357:455–460 (1992); (2) as direct oncolytic agents by using viruses that have been genetically modified to lose their pathogenic features, Martuza, R. L., et al., Science, 252:854–856 (1991); or (3) as agents to selectively damage malignant cells using viruses which have been genetic engineered for this purpose, Bischoff, J. R., et al., Science, 274:373–376 (1996).
Examples for the use of viruses against malignant gliomas include the following.
Herpes Simplex Virus dlsptk (HSVdlsptk), is a thymidine kinase (TK)-negative mutant of HSV. This virus is attenuated for neurovirulence because of a 360-base-pair deletion in the TK gene, the product of which is necessary for normal viral replication. It has been found that HSVdlsptk retains propagation potential in rapidly dividing malignant cells, causing cell lysis and death. Unfortunately, all defective herpes viruses with attenuated neuropathogenicity have been linked with serious symptoms of encephalitis in experimental animals. Wood, M. J. A., et al., Gene Therapy, 1:283–291 (1994). For example, in mice infected intracerebrally with HSVdlsptk, the LD50Ic (intracranial administration) is 106 pfu, a rather low dose. This limits the use of this mutant HSV. Markert, J. M., et al., Neurosurgery, 32:597–603 (1993). Other mutants of HSV have been proposed and tested. Nevertheless, death from viral encephalitis remains a problem. Mineta T., et al., Nature Medicine, 1:938–943 (1995); Andreansky, S., et al., Cancer Res., 57:1502–1509 (1997).
Another proposal is to use retroviruses engineered to contain the HSV tk gene to express thymidine kinase which causes in vivo phosphorylation of nucleoside analogs, such as gancyclovir or acyclovir, blocking the replication of DNA and selectively killing the dividing cell. Izquierdo, M., et al., Gene Therapy, 2:66–69 (1995) reported the use of Moloney Murine Leukemia Virus (MoMLV) engineered with an insertion of the HSV tk gene with its own promoter. Follow-up of patients with glioblastomas that were treated with intraneoplastic inoculations of therapeutic retroviruses by MRI revealed shrinkage of tumors with no apparent short-term side effects. However, the experimental therapy had no effect on short-term or long-term survival of affected patients. Retroviral therapy is typically associated with the danger of serious long-term side effects (e.g. insertional mutagenesis).
Chen, S. H., et al., PNAS, USA, 91:3054–3057 (1994) reported the direct injection of a recombinant into experimentally induced gliomas in athymic mice. ADV/RSV-TK is an adenovirus containing the HSV-tk gene under transcriptional control of the rous sarcoma virus long terminal repeat, followed by treatment with gancyclovir. The treatment caused tumor necrosis without apparent involvement of the cellular immune response. The treated animals survived >50 days after tumor inoculation as contrasted with control tumor inoculated animals all of which died after 23 days. However, further long-term toxicity testing of neuronal, glial and endothelial cells is necessary to assess the potential of genetically engineered retroviruses for the treatment of cancers.
Recently, a novel strategy to use human pathogenic viruses for the treatment of malignant disease was introduced. Adenovirus engineered to selectively replicate within and destroy malignant cells expressing a modified p53 tumor suppressor offers an opportunity to target malignant cells without causing unwanted side effects due to virus propagation at extratumoral sites. Bischoff, J. R., et al., supra.
Similar systems have been developed to target malignancies of the upper airways, tumors that originate within the tissue naturally susceptible to adenovirus infection and that are easy accessible. However, Glioblastoma multiforme, highly malignant tumors composed out of widely heterogeneous cell types (hence the denomination multIforme) are characterized by exceedingly variable genotypes and are unlikely to respond to oncolytic virus systems directed against homogeneous tumors with uniform genetic abnormalities.
The Cells of the Central Nervous System
It is important to recognize that there are two classes of cells in the brain, the neural cells (neurons) and the neuroglia cells (glia). Neurons process information received from the peripheral receptors giving rise to perception and memory. Motor commands are issued and transmitted also by means of neurons to the various muscles of the body. There are nine times more glial cells than neurons. The glial cells have multiple functions. They serve as the supporting elements; segregate neurons into disparate groups and produce myelin. Based on physiological characteristics, there are five major classes of glial cells: astrocytes, oligodendrocytes, microglia, ependymal cells, and Schwann cells. Kandel, E. R. and Schwartz, J. H., ed., Principles of Neural Science, Chapter 2, pp. 14–23 Elsevier/North, Holland, 1981.
It is known that both the neurons and glial cells emerge from the neuroepithelium of the primitive neural tube. However, the timing and place of the mechanisms that underlie the separation of neuronal and glial cell lines have been unsettled and controversial. In 1889, His proposed that the germinative epithelium consists of two classes of precursor cells: one that produces neurons and another that produces glial cells. Although disputed, this has proven to be correct. It is believed that glial cells are generated after all or a majority of the neurons destined for a given structure have been formed. Black, I., ed. Cellular and Molecular Biology of Neuronal Development, Chapter 2, pp. 29–47, Plenum Press, New York, 1984.
The Poliovirus
Poliomyelitis is a disease of the central nervous system caused by infection with poliovirus. Poliovirus is a human enterovirus that belongs to the PIcornavIrIdae family and is classified into three stable serotypes. It is spherical, 20 nm in size, and contains a core of RNA coated with a capsule consisting of proteins. It is transmitted through the mucosa of the mouth, throat or the alimentary canal. All three poliovirus serotypes have been reported as causative agents of paralytic poliomyelitis, albeit at different frequencies (type 1>type 2>type 3).
However, infection by poliovirus does not necessarily lead to the development of poliomyelitis. On the contrary, the majority of infections (98–99%) lead to local gastrointestinal replication of the virus causing only mild symptoms, or no symptoms at all. Rarely does poliovirus invade the CNS where it selectively targets spinal cord anterior horn and medullary motor neurons for destruction. Bodian, D., in: Diseases of the Nervous System, Minckler, J. ed., McGraw-Hill, New York, pp. 2323–2339 (1972).
The unusually restricted cell tropism of poliovirus leads to unique pathognomonic features. They are characterized by motor neuron loss in the spinal cord and the medulla, giving rise to the hallmark clinical sign of poliomyelitis, flaccid paralysis. Other neuronal components of the central nervous system as well as glial cells typically escape infection. In infected brain tissue under the electronmicroscope, severe changes are observed in motor neurons whereas no significant alterations are observed in the neuroglial components. Normal astrocyes and oligodendrocytes may be seen next to degenerate neurons or axons without evidence of infection or reaction. Bodian, D., supra. The restricted tropism of poliovirus is not understood. In addition to the restricted cell and tissue tropism, poliovirus only infects primates and primate cell cultures. Other mammalian species remain unaffected. Ren, R., et al., Cell, 63:353–362 (1990).
The isolation of poliovirus in 1908 led to intensive research efforts to understand the mechanisms of infection. The earlier work required the use of monkeys and chimpanzees as animal models. Such animals with longer life cycles are very costly and difficult to use in research. The discovery of the human poliovirus receptor (PVR) also known as CD155, the cellular docking molecule for poliovirus, led to the development of a transgenic mouse expressing the human poliovirus receptor as a new animal model for poliomyelitis. The pathogenicity of poliovirus may be studied using the transgenic mice. Ren et al. (1990); Koike, S., et al., PNAS, USA, 88:951–955 (1991).
The early research efforts have also led to the development of attenuated PV strains that lack neuropathogenic potential and soon were tested as potential vaccine candidates for the prevention of poliomyelitis. The most effective of these are the Sabin strains of type 1, 2, and 3, of poliovirus developed by A. Sabin. Sabin & Boulger., Dev. Biol. Stand. 1:115–118 (1973). After oral administration of the live attenuated strains of poliovirus (the Sabin strains) vaccine-associated paralytic poliomyelitis has been observed in extremely rare cases. The occurrence of vaccine-associated paralytic polio has been correlated with the emergence of neurovirulent variants of the attenuated Sabin strains after immunization. Minor, P. D., Dev. Biol. Stand., 78:17–26 (1993).
In order to understand the invention, it is important also to have an understanding of the structure of poliovirus.
All picornaviruses including enteroviruses, cardioviruses, rhinoviruses, aphthoviruses, hepatovirus and parechoviruses contain 60 copies each of four polypeptide chains: VP1, VP2, VP3, and VP4. These chains are elements of protein subunits called mature “protomers”. The protomer is defined as the smallest identical subunit of the virus. Traces of a fifth protein, VP0, which is cleaved to VP2 and VP4 are also observed. Together, these proteins form the shell or coat of poliovirus.
The picornaviral genome consists of a single strand of messenger-active RNA. The genomic messenger active RNA consists of a “+” strand which is polyadenylated at the 3′ terminus and carries a small protein, VPg, covalently attached to the 5′ end. The first picornaviral RNA to be completely sequenced and cloned into DNA was that of a type 1 poliovirus. However, polioviruses lack a 5′m7GpppG cap structure, and the efficient translation of RNA requires ribosomal binding that is accomplished through an internal ribosomal entry site (IRES) within the 5′ untranslated region (5′NTR).
The common organizational pattern of a poliovirus is represented schematically in FIG. 1, which comprises 5′NTR, P1, P2, P3 and 3′NTR with a polyadenylated tail. The 5′NTR comprises 6 domains arbitrarily designated as I, II, III, IV, V, and VI. The IRES comprises domains II–VI. P1 is the coding region for structural proteins also known as the capsid proteins. P2 and P3 encode the non-structural proteins. A schematic diagram of the six domains of the 5′NTR is represented in FIG. 2.
In nature, three immunologically distinct poliovirus types occur: serotype 1, 2, and 3. These types are distinct by specific sequences in their capsid proteins that interact with specific sets of neutralizing antibodies. All three types occur in different strains, and all naturally occurring types and strains can cause poliomyelitis. They are, thus, neurovirulent. The genetic organization and the mechanism of replication of the serotypes are identical; the nucleotide sequences of their genomes are >90% identical. Moreover, all polioviruses, even the attenuated vaccine strains, use the same cellular receptor (CD155) to enter and infect the host cells; and they express the same tropism for tissues in human and susceptible transgenic animals.
The neuropathogenicity of poliovirus can be attenuated by mutations in the regions specifying the P1 and P3 proteins as well as in the internal ribosomal entry site (IRES) within the 5′NTR. The Sabin vaccine strains of type 1, 2, and 3 carry a single mutation each in domain V of their IRES elements that has been implicated in the attenuation phenotype. Despite their effectiveness as vaccines, the Sabin strains retain a neuropathogenic potential in animal models for poliomyelitis. Albeit at a very low rate, they can cause the disease in vaccinees.
Indeed, the single point mutations in the IRES element of each Sabin vaccine strain can revert in a vaccinee within a period of 36 hours to several days. Overall, vaccine associated acute poliomyelitis occurs in the United States at a rate of 1 in 530,000 vaccinees. The polioviruses isolated from vaccinated patients with poliomyelitis may also have mutations reverted in different positions of their genomes. Wimmer, E., et al., Ann.Rev.Gen., 27:353–436 (1993), Minor, P. D., supra.
Recombinant Polioviruses
Chimeric polioviruses carrying heterologus IRES elements, which have lost their inherent neuropathogenic potential have been described. Gromeier, M., et al., Proc. Natl. Acad. Sci. USA, 93:2370–2375 (1996), incorporated herein by reference. It was found that the substitution of the cognate IRES of poliovirus with its counterpart from Human Rhinovirus type 2 (HRV2) eliminated the ability of the resulting chimera, PV1(RIPO) to grow within cells of neuronal derivation (FIGS. 3A and B). The inventors also described the construction of and neurovirulence testing of a chimera carrying the P1 coding region for the structural proteins derived from PV1(S), PV1(RIPOS) in addition to the heterologous IRES originating from HRV2.
The non-pathogenic phenotype of PV1(RIPO) and PV1(RIPOS) was documented in mice transgenic for the human poliovirus receptor, CD155 tg mice. See Gromeier et al., supra. It was shown that non-pathogenic PV/HRV2 IRES chimeras are unable to cause the typical lesions of the spinal cord typical of poliomyelitis when injected intracerebrally into CD155 tg mice. The non-pathogenic property of these constructs are now shown in Cynomolgus monkeys (FIG. 3A). The non-human primates that received intraspinal inoculations of PV1(RIPO) or PV1(RIPOS) remained unaffected or developed transient, subtle pareses of one foot in an isolated case. Permanent neurological dysfunction or signs of poliomyelitic disease were not noticed in any of the treated monkeys.
Despite its inability to replicate efficiently within cells of neuronal origin, it is now shown that PV1(RIPO) retained wild-type growth characteristics with an ability to lyse tumor cells in a panel of rapidly dividing malignant cell types originating from human malignancies (FIGS. 10–17).